This invention relates to methods, compositions and articles for assaying a sample for an analyte.
A colorless, odorless, tasteless chemical has become one of the most dangerous illicit drugs of abuse today. The drug is a central nervous system (CNS) depressant at low doses, and has the curious effects of reducing anxiety and producing euphoria and relaxation, sedating the recipient. The drug also is naturally present in the body and has a short half-life, making detection of ingested drug difficult (S. D. Ferrara et al., Journal of Pharmacology and Biomedical Analysis, 11:6, 483-487, 1993).
Because of these properties, the drug has been abused through surreptitious administration to unsuspecting users in a variety of settings, including college parties and bars. The drug has thus become known as one of the xe2x80x9cdate rapexe2x80x9d drugs, used to disable women who have unknowingly ingested the drug in a product they otherwise intended to consume.
The drug has risks beyond unintended disinhibition, however. The drug can cause unconsciousness, respiratory depression, bradycardia, nausea, vomiting, seizures and coma, and has been linked to over 60 deaths. The severity of symptoms and the duration of action are dose dependent and can be affected by the presence of other CNS depressants. Recently, male perpetrators who mixed the drug in the drinks of unsuspecting females were convicted of involuntary manslaughter in the death of one 15-year-old recipient who died as a result (xe2x80x9cJustice, with tears,xe2x80x9d Schmitt et al., Detroit Free Press, Mar. 15, 2000).
The dangers of such results are an inherent property of the drug, and one that makes its illicit use particularly dangerous. The drug has a very steep dose-response curve. A 1-gram dose for a 150-pound person provides a low degree of effect, causing a sense of euphoria and loss of inhibitions (Leonard, 1999; Galloway, 2000). However, a 2.5-gram dose to the same individual can lead to coma (ibid.). Higher doses can result in death.
Despite the rapid rise in the degree of abuse of this drug, the available methods for assaying GHB are either insensitive or cumbersome, difficult, time-consuming, expensive and equipment-intensive (J. Letteri and H. Fung, Journal of Pharmacology and Experimental Therapeutics, 208, 7-11, 1979). Given the short half-life of the compound and the public settings in which it is used and abused, it is currently difficult at best to assay for its presence, and cannot be done routinely and practically in public venues where it is most often abused, such as bars. The time-consuming nature or insensitivity of the available assays also impedes rapid treatment of overdose victims, particularly when they are unconscious or otherwise unaware of having ingested anything, because proper therapy is dictated by knowledge of the kind of overdose being treated.
The drug is gamma-hydroxybutyric acid (GHB). GHB was widely used for a number of years as a freely available, over the counter supplement. Bodybuilders used GHB for its reported effects of inducing the release of growth hormone from the anterior pituitary. GHB has a number of potentially useful therapeutic properties, and has been used as an anesthetic and in the treatment of insomnia, narcolepsy, drug addiction, and withdrawal symptoms. GHB has been suggested to be a natural neurotransmitter; receptors for it have been detected in the brain, and mechanisms for the synthesis, release and uptake of GHB in the brain have been characterized. GHB is also chemically related to the brain""s major inhibitory neurotransmitter, gamma-aminobutyric acid (GABA).
Although GHB was previously readily available, abuse has led to its sale and use being highly restricted. Hospital emergency room visits resulting from the use of GHB increased from 55 in 1994 to 2,973 in 1999 (The DAWN Report, December 2000, Substance Abuse and Mental Health Services Administration). The dangers from the drug were thought to be so high that a federal law was enacted identifying GHB as an imminent hazard to public safety and directing the Attorney General to classify GHB as a xe2x80x9cSchedule 1xe2x80x9d drug under the Controlled Substances Act, increasing the penalties for its illicit use (H.R. 2130/S. 1561, the Hillory J. Farias and Samantha Reid Date-Rape Drug Prohibition Act of 2000xe2x80x2). Unfortunately, metabolic precursors to GHB such as gamma-butyrolactone (GBL) and 1,4-butanediol remain readily available, and are subject to the same forms of abuse.
Despite the serious recognized problems presented by GHB use, until very recently there has been no field test available that allows the detection of GHB under real-world conditions that can address the current methods of abuse. Known methods of GHB detection are laborious, requiring multiple steps, expensive instrumentation, and trained technicians to perform them, or they are too insensitive to detect GHB at concentrations commonly abused. While existing methods could be used to detect GHB in forensic samples after a victim or suspect has already been identified, they cannot practically be applied routinely on multiple samples in the field prior to ingestion. A recently developed chemical field method is very insensitive and does not reliably detect the concentration of GHB typically present in adulterated drinks.
For example, gas chromatography-mass spectrometry methods for detecting GHB have been described (McCusker et al., Journal of Analytical Toxicology Sep. 23, 1999(5):301-5; Gibson et al., Biomed. and Environ. Mass Spectrometry, 19, 89-93, 1990). Gas chromatographic methods may not detect GHB directly, as GHB can be converted to the lactone form at injector temperatures. Additionally, detection methods can involve acidification steps coupled with extraction and/or gas chromatography; such acidification converts any GHB present to GBL. This is problematic, as the lactone form is not illegal per se. A method for detecting GHB that avoids detection of gamma-butyrolactone was discussed in U.S. Pat. No. 6,156,431.
HPLC methods of detecting GHB were described by M. Z. Mesmer and R. D. Satzger (J. of Forensic Sci., 43(3), 489-492, May, 1998). Both methods involve reverse phase HPLC, which is coupled with UV detection at 215 nm in the first method, and with thermospray mass spectrometry in the second. These methods can resolve GHB and GBL via HPLC. Both methods require expensive laboratory equipment and a trained laboratory technician to operate them. Furthermore, the methods may lack the sensitivity needed to detect the compound(s) in dilute samples such as adulterated drinks or metabolic fluids. The first method additionally requires a sample lacking UV absorbing substances.
The Scott Company (133 Red Oak Lane, Flower Mound, Tex. 75128; 972-539-0229) recently introduced a chemical color test for GHB. A Q-tip(copyright) is soaked in the solution to be tested and placed in a small vial containing a yellow solution. A change in color from yellow to brown indicates the presence of GHB. Analysis of different GHB concentrations using the Scott kit demonstrated that 300 mM GHB gives a weak positive test. This is a very high concentration of GHB that in a 4-ounce drink could cause death. GHB commonly is abused at lower concentrations that yield negative tests with the Scott kit. Also, the Scott test does not detect precursors to GHB.
There is a need in the art for new methods of analyzing GHB in a sample, and for devices, compositions and articles of manufacture useful in such methods.
Methods, compositions and articles for enzymatically assaying a sample for a GHB source are provided. The methods advantageously employ a first oxidoreductase that can oxidize 4-hydroxybutyric acid. Conversion of this acid to succinic semialdehyde through the enzymatic activity of the oxidoreductase is coupled to the reduction of a cofactor for the first oxidoreductase. The reduced form of this cofactor can be assayed directly, for example spectrophotometrically to detect changes in absorbance of the cofactor following reduction. Or additional enzymatic methods may be employed which utilize the reduced cofactor to produce a detectable signal.
Preferably, a hydride abstractor is used which abstracts a hydride from the reduced cofactor and produces a detectable change. The detectable change can occur in the hydride abstractor or in another molecule. Preferably, a second oxidoreductase is employed as the hydride abstractor, and the detectable change occurs in a chromogen or dye that is reduced by the second oxidoreductase in concert with oxidation of the reduced cofactor. Where the method is performed as a field test outside a laboratory setting, the detectable change is preferably a visually detectable change in the chromogen or dye which permits assay results to be visually determined.
The methods can be used in solution or can take place on or within a support, for example on a test strip. Positive controls containing known GHB sources may also be employed. When performed on a support such as a test strip, the positive control(s) may be deposited in a detectable pattern to allow for easier detection of a positive result. Reagents for performing the assay on the test sample can also be deposited on the support, providing a defined area in which a positive result is determined. Different chromogens and/or dyes can be used which produce different detectable changes, for example different color changes, to allow for use of the assay on a samples of various colors, and may be fixed on different regions of the support.
The methods can be employed on a single sample or on multiple samples, for example in a multiwell or other array format. The methods can be used to detect a GHB source in a sample, and can also be used to quantitate the amount of a GHB source present in the sample.
The methods can incorporate additional techniques to detect precursor sources of GHB. For example, an esterase may be included in the assay to allow detection of esterified forms of GHB, including the internal ester gamma-butyrolactone. Comparison of assay results in the presence and absence of such an esterase allows the method to distinguish between GHB and esterified forms in the sample. The methods can also employ steps for altering or removing ethanol from the sample to prevent a false positive result from occurring where the first oxidoreductase can use ethanol as an alternative substrate.
Compositions comprising reagents useful for performing the assay are also provided. Kits comprising reagents useful for performing the methods of the invention are also provided. The methods, compositions and articles can be used as alternatives to other methods of assaying samples for GHB.